GSM7977735: Seedling, 57h light, rep2, ATAC-seq; Arabidopsis thaliana; ATAC-seq
Germinating seeds were placed between two microscope slides with a small drop of water which were rotated and squeezed together with gentle force until the embryos were released from within the seed. The mixture of embryos and seed coats were then kept in a small beaker containing 4 mL ice-cold water until all of the sample had been processed (1-2 g starting material). Following this, 4 mL 80% sucrose was added and mixed gently by inversion. The solution was split into two 5 mL microcentrifuge tubes and centrifuged at 3,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The embryo-containing supernatant was filtered through a piece of miracloth (EMD Millipore), washed with ice-cold water, and briefly dried from below using a piece of paper towel to absorb excess water. The embryos were flash frozen in a mortar and ground to a fine powder with a pestle. Approximately 500 mg of frozen ground tissue powder was added to 10.5 mL ice-cold nuclear purification buffer (NPB; 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine) with added cOmplete Mini EDTA-free protease inhibitor cocktail (Merck) in a chilled mortar and ground with a chilled pestle. The homogenate was collected with a 10 mL serological pipette and passed through a 100 µm cell strainer, as well as a 40 µm cell strainer, into a 15 mL Falcon tube (Corning) kept on ice. The tube was centrifuged at 1,200 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The supernatant was discarded and the pellet gently resuspended in 1 mL ice-cold nuclear extraction buffer 2 (NEB2; 0.25 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl2, 1% Triton X-100) with added cOmplete Mini EDTA-free protease inhibitor cocktail (Merck) and transferred to a cooled 1.5 mL microcentrifuge tube. The tube was centrifuged at 12,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The supernatant was removed and the pellet resuspended in 300 µL ice-cold NEB2. This was then pipetted as a layer above 300 µL of nuclear extraction buffer 3 (NEB3; 1.7 M sucrose, 10 mM Tris-HCl pH 8, 2 mM MgCl2, 0.15% Triton X-100) with added cOmplete Mini EDTA-free protease inhibitor cocktail (Merck) in a new 1.5 mL microcentrifuge tube. The tube was centrifuged at 16,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The supernatant was removed, the pellet resuspended in 500 µL ice-cold NPB and transferred to a new 1.5 mL microcentrifuge tube. Nuclei concentration and quality was determined using fluorescence microscopy. Briefly, 1 µL DAPI (0.2 µg/µL) was added to 25 µL nuclei in a 0.5 mL microcentrifuge tube and kept in the dark on ice. A fluorescence microscope was used to count nuclei from 10 µL of the DAPI-stained nuclei inside a Neubauer Improved chamber (Merck). Tagmentation and library preparation was performed following the manufacturers instructions in the Diagenode Tagmentation kit (Diagenode). Approximately 50,000 nuclei were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 1,000 x g for 10 min in a cooled benchtop centrifuge (4 ˚C). The nuclei pellet was gently resuspended in 50 µL tagmentation mastermix (25 µL 2X Tagmentation buffer (Diagenode), 16.5 µL PBS pH 7.4, 5 µL nuclease-free water, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, 2.5 µ